EGF (Epidermal growth factor) is a polypeptide consisting of 53 amino acids, and first identified to be secreted from the submandibular gland of rats. Since then, in addition to the ability to stimulate the growth of skin cells of epithelium and epidermis in various tissues of the body, hEGF (human epidermal growth factor) has been known to promote the growth and differentiation of epithelial cells of the cornea and lung (Carpenter, G. Handbook of Experimental Pharmacology, 1981, 57, 89). Due to these features, the EGF has been developed and widely used as the wound medicine, and recently, is drawing attention as the raw material of functional cosmetics with proven anti-wrinkle and anti-aging effects (Brown, G. L, U.S. Pat. No. 5,618,544). Initially, EGF has limitation in productivity due to being extracted from human placenta and urine, but the productivity has been improved enough to be used as a cosmetic raw material as the recombinant hEGF production systems have been developed due to the development of genetic engineering techniques. Demand of hEGF is gradually increasing according to extending the scope of use, but hEGF has become a major factor raising the price of functional cosmetics due to the high production cost. Thus, in order to popularize functional cosmetics and expand the market of hEGF products, the issue of hEGF production cost must be solved.
The recombinant expression of the desired protein is widely used as a method for mass production of proteins for research, therapeutic, or other commercial purposes. In order to produce large quantities of recombinant proteins, various vectors and hosts are used, and typically a method using E. coli is widely used. Most of currently produced hEGF is produced by expressing a synthetic human EGF gene in E. coli, and the case of expression in yeast has been reported (Urdea, N. S. et al. P.N.A.S., 1983, 86, 7461; Korea Patent 1990-0022194). When expressed in E. coli, the gene is expressed in the form of fusion protein in combination with fibronectin collagen-binding domain (Ishikawa, T. et al., J. Biochem., 2001, 129, 627), amino terminus of swine growth factor (Xial, C J et al., J. Mol. Endocrinol., 1996, 16, 89) or a part of TrpE protein of E. coli (Allen, G, et al., J. Cell Sci. Suppl., 1985, 3, 29), or expressed simply by adding six histidine amino acids (Lee, J Y et al. Biotechnol. Appl. Biochem., 2000, 31, 245).
These methods provide a high protein expression level, but have the disadvantage of high cost due to necessities of the activation process in order to form the tertiary structure consisting of three cysteine bonds such as hEGF protein and activate to form a three-dimensional structure since the protein is expressed in the form of inactive inclusion body, and various stages of the purification process in order to obtain the hEGF with high purity.
Meanwhile, yeast Saccharomyces cerevisiae, which is a eukaryotic microorganism, is a GRAS (Generally Recognized As Safe) microorganism proven safe for humans, easy to modify genetically, and has various expression systems developed. In addition, it provides the advantages of performing the secretory function to secrete the proteins out of the cell when recombining the higher cell-derived proteins such as human proteins and post-translational modification of protein such as glycosylation. In order to produce the target protein through secretion, extracellular secretion is possible by artificial fusion of protein secretory signal and the target protein, and protein folding, disulfide bond formation and the addition process of sugar chain are progressing through the secretion process of protein. Therefore, it offers the advantage of producing the recombinant protein with biologically complete activity. It also can recover the biologically active proteins directly from the medium without breaking the cells, and is very economical because the refolding step is not required (Eckart and Bussineau, Curr. Opin. Biotechnol., 1996, 7, 525).
However, due to low productivity of the active form of EGF compared to the E. coli, the yeast Saccharomyces cerevisiae has not received attention as EGF-producing strain.
Accordingly, as the results of the efforts to produce the active hEGF in yeast with high concentration and high purity, the present inventors identified that, if expressing hEGF fused with HL (Hydrophilic domain) peptide having a specific amino acid sequence derived from VOA1 gene in KEX1 gene-deleted strains, hEGF with the same conformation and activity as wild type can be mass-produced, to complete the present invention.